Prevalence and treatment of gastric ulcers in Thoroughbred racehorses of Korea.

BACKGROUND: Gastric ulcer is one of the prevalent diseases in racehorses. However, it has not been recognized as important in Korea, and drugs used to treat gastric ulcers are included in the doping test list, so they are not allowed to be administered to racehorses in training. OBJECTIVES: This study was performed 1) to investigate the prevalence and the severity of gastric ulcers in Thoroughbred racehorses in Korea, 2) to confirm the therapeutic effect of ranitidine and omeprazole, and 3) to compare the efficacy between ranitidine and omeprazole. METHODS: Forty-nine horses were randomly recruited, and gastroscopy was performed within two days after racing. Twelve horses with a sum grade of five or higher were randomly assigned to two treatment groups. Seven horses were administered ranitidine, and five horses were administered omeprazole. Follow-up gastroscopy was scheduled within one to five days after finishing the treatment. RESULTS: The prevalence of gastric ulcer in Korean Thoroughbred racehorses after racing was 100%, and the grade was more severe in the non-glandular region than in the pyloric region. There was no correlation between the severity of gastric ulcer in the two regions. Omeprazole had a greater therapeutic effect than ranitidine. CONCLUSIONS: This study shows the importance of recognizing gastric ulcers as an important factor, and omeprazole as a possible treatment option in Korea, as it has been removed from the list of prohibited substances for racehorses. Thus, the use of omeprazole is currently recommended until one day before the race.

Single-nucleotide polymorphism typing analysis for molecular subtyping of Salmonella Tennessee isolates associated with the 2007 nationwide peanut butter outbreak in the United States.

BACKGROUND: In 2007, a nationwide Salmonella Tennessee outbreak occurred via contaminated peanut butter. Here, we developed a single-nucleotide polymorphism (SNP)-typing method for S. Tennessee to determine the clonal subtypes of S. Tennessee that were associated with the peanut butter outbreak. METHODS AND RESULTS: One seventy-six S. Tennessee isolates from various sources, including humans, animals, food, and the environment, were analyzed by using the SNP technique. Eighty-four representative SNP markers were selected by comparing the sequences of three representative S. Tennessee strains with different multi-locus sequence typing and variable number tandem repeats from our collection. The set of eighty-four SNP markers showed 100% typeability for the 176 strains, with the nucleotide diversity ranging from 0.011 to 0.107 (mean = 0.049 ± 0.018, median = 0.044) for each marker. Among the four clades and nine subtypes generated by the SNP typing, subtype 1, which comprised 142 S. Tennessee strains, was the most predominant. The dominance of single-strain clones in subtype 1 revealed that S. Tennessee is highly clonal regardless of outbreak-association, source, or period of isolation, suggesting the presence of an S. Tennessee strain prototype. Notably, a minimum 18 SNP set was able to determine clonal S. Tennessee strains with similar discrimination power, potentially allowing more rapid and economic strain genotyping for both outbreaks and sporadic cases. CONCLUSIONS: The SNP-typing method described here might aid the investigation of the epidemiology and microevolution of pathogenic bacteria by discriminating between outbreak-related and sporadic clinical cases. In addition, this approach enables us to understand the population structure of the bacterial subtypes involved in the outbreak.

Prevalence, virulence potential, and pulsed-field gel electrophoresis profiling of Shiga toxin-producing Escherichia coli strains from cattle.

BACKGROUND: As a primary source of Shiga-toxin-producing Escherichia coli (STEC) infection, cattle are often targeted to develop strategies for reducing STEC contamination. Monitoring the virulence potentials of STEC isolates from cattle is important for tracing contamination sources, managing outbreaks or sporadic cases, and reducing the risks for human infection. This study aimed to investigate the prevalence of STEC in cattle farm samples in South Korea and to assess their virulence potentials. RESULTS: In total, 63 STEC were isolated from 496 cattle farm samples, and temperature and rainfall affected STEC prevalence (p < 0.001). The O157 serogroup was most prevalent, followed by O108, O8, O84, O15, and O119. In the stx variant test, high prevalence of stx2a and stx2c (known to be associated with high STEC virulence) were observed, and stx2g, a bovine STEC variant, was detected in STEC O15 and O109. Additionally, stx1c was detected in eae-positive STEC, suggesting genetic dynamics among the virulence genes in the STEC isolates. STEC non-O157 strains were resistant to tetracycline (17.9%), ampicillin (14.3%), and cefotaxime (3.6%), while STEC O157 was susceptible to all tested antimicrobials, except cefotaxime. The antimicrobial resistance genes, blaTEM (17.5%), tetB (6.3%), and tetC (4.8%), were only detected in STEC non-O157, whereas tetE (54.0%) was detected in STEC O157. AmpC was detected in all STEC isolates. Clustering was performed based on the virulence gene profiles, which grouped STEC O84, O108, O111, and O157 together as potentially pathogenic STEC strains. Finally, PFGE suggested the presence of a prototype STEC that continues to evolve by genetic mutation and causes within- and between-farm transmission within the Gyeonggi province. CONCLUSIONS: Considerable numbers of STEC non-O157 were isolated from cattle farms, and the virulence and antimicrobial resistance features were different between the STEC O157 and non-O157 strains. STEC from cattle with virulence or antimicrobial resistance genes might represent a threat to public health and therefore, continual surveillance of both STEC O157 and non-O157 would be beneficial for controlling and preventing STEC-related illness.

Antibiotic resistance patterns and genetic relatedness of Enterococcus faecalis and Enterococcus faecium isolated from military working dogs in Korea.

Enterococcus spp. are normally present in the gastrointestinal tracts of animals and humans, but can cause opportunistic infections that can be transmitted to other animals or humans with integrated antibiotic resistance. To investigate if this is a potential risk in military working dogs (MWDs), we analyzed antibiotic resistance patterns and genetic relatedness of Enterococcus spp. isolated from fecal samples of MWDs of four different age groups. Isolation rates of Enterococcus spp., Enterococcus (E.) faecalis, and E. faecium, were 87.7% (57/65), 59.6% (34/57), and 56.1% (32/57), respectively, as determined by bacterial culture and multiplex PCR. The isolation rate of E. faecalis gradually decreased with age (puppy, 100%; adolescent, 91.7%; adult, 36.4%; and senior, 14.3%). Rates of resistance to the antibiotics ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole/trimethoprim, imipenem, and kanamycin among Enterococcus spp. increased in adolescents and adults and decreased in senior dogs, with some isolates having three different antibiotic resistance patterns. There were indistinguishable pulsed-field gel electrophoresis patterns among the age groups. The results suggest that Enterococcus is horizontally transferred, regardless of age. As such, periodic surveillance studies should be undertaken to monitor changes in antibiotic resistance, which may necessitate modification of antibiotic regimens to manage antibiotic resistance transmission.

The Fecal Microbial Communities of Dairy Cattle Shedding Shiga Toxin-Producing Escherichia coli or Campylobacter jejuni.

Cattle are a natural reservoir of Shiga toxin-producing Escherichia coli (STEC) and have recently been recognized as a major source of Campylobacter jejuni contamination. While several factors are known to be associated with bacterial colonization, the underlying microbial factors have not been clarified. In this study, we characterized the fecal microbiota of dairy cattle (n = 24) using next-generation sequencing to elucidate the intestinal bacterial communities and the microbial diversity in relation to the presence of the foodborne pathogens STEC and C. jejuni (STEC-positive samples, n = 9; STEC-negative samples, n = 15; C. jejuni-positive samples, n = 9; and C. jejuni-negative samples, n = 15). While no significant differences were observed in alpha diversity between STEC-positive and STEC-negative samples, a high diversity index was observed in C. jejuni-positive samples compared to C. jejuni-negative samples. Nine phyla, 13 classes, 18 orders, 47 families, 148 genera, and 261 species were found to be the core microbiota in dairy cattle, covering 80.0-100.0% of the fecal microbial community. Diverse microbial communities were observed between cattle shedding foodborne pathogens and nonshedding cattle. C. jejuni-positive cattle had a higher relative abundance of Bacteroidetes (p = 0.035) and a lower relative abundance of Firmicutes (p = 0.035) compared to C. jejuni-negative cattle. In addition, while the relative abundance of 2 and 6 genera was significantly higher in cattle-shedding STEC and C. jejuni, respectively, the relative abundance of 3 genera was lower in both STEC- and C. jejuni-negative cattle. Our findings provide fundamental information on the bacterial ecology in cattle feces and might be useful in developing strategies to reduce STEC or C. jejuni shedding in dairy cattle, thereby reducing the incidence of STEC infection and campylobacteriosis in humans.

Diversity of the Gastric Microbiota in Thoroughbred Racehorses Having Gastric Ulcer.

Equine gastric ulcer syndrome is one of the most frequently reported diseases in thoroughbred racehorses. Although several risk factors for the development of gastric ulcers have been widely studied, investigation of microbiological factors has been limited. In this study, the presence of Helicobacter spp. and the gastric microbial communities of thoroughbred racehorses having mild to severe gastric ulcers were investigated. Although Helicobacter spp. were not detected using culture and PCR techniques from 52 gastric biopsies and 52 fecal samples, the genomic sequences of H. pylori and H. ganmani were detected using nextgeneration sequencing techniques from 2 out of 10 representative gastric samples. The gastric microbiota of horses was mainly composed of Firmicutes (50.0%), Proteobacteria (18.7%), Bacteroidetes (14.4%), and Actinobacteria (9.7%), but the proportion of each phylum varied among samples. There was no major difference in microbial composition among samples having mild to severe gastric ulcers. Using phylogenetic analysis, three distinct clusters were observed, and one cluster differed from the other two clusters in the frequency of feeding, amount of water consumption, and type of bedding. To the best of our knowledge, this is the first study to investigate the gastric microbiota of thoroughbred racehorses having gastric ulcer and to evaluate the microbial diversity in relation to the severity of gastric ulcer and management factors. This study is important for further exploration of the gastric microbiota in racehorses and is ultimately applicable to improving animal and human health.

Helicobacter apodemus sp. nov., a new Helicobacter species identified from the gastrointestinal tract of striped field mice in Korea.

A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers AF284754, AY009129, and AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.

Development of a loop-mediated isothermal amplification assay for rapid, sensitive detection of Campylobacter jejuni in cattle farm samples.

Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuni and 41 non-C. jejuni strains, respectively), sensitive (detection limit of 100 fg/µl), and quantifiable (R(2) = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean ~10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis.

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle.

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on Tm values of 85.03 ± 0.54°C for stx1 and 87.47 ± 0.35°C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R² = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.

Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA.

Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07℃ for cattle, 84.96±0.08℃ for pig, and 85.99±0.05℃ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11℃ for goat and 83.90±0.11℃ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23℃ for chicken, 88.66±0.12℃ for duck, and 84.49±0.08℃ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/µL to 100 fg/µL levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.